Project Summary The proposed work will address novel mechanisms of regulation of cell migration, building on two fundamental paradigms that have been established through my independent work since 2004. First, I demonstrated the essential biological role of protein arginylation and demonstrated that this posttranslational modification regulates multiple proteins in vivo and critically affects cell migration. Second, I discovered that during cell migration the coding sequence, rather than the amino acid sequence, drives functional distinction between homologous protein isoforms by regulating their rates of translation, posttranslational modifications, and function. This work, published in a number of high impact journals (including 18 peer reviewed publications in the last 5 years) have established my lab as a leader in the field and the group uniquely suited to perform the proposed studies. We will combine these two innovative concepts into an integrated study that will uncover novel mechanisms of the regulation of cell migration by mRNA coding sequence and arginylation. We hypothesize that: (1) mRNA- mediated regulation of N-terminal arginylation of beta actin uniquely regulates actin function during cell migration by facilitating actin polymerization at the cell leading edge; (2) ATE1's activity and targeting to specific protein substrates and sites is regulated locally at the leading edge and/or globally throughout the cell at the onset of cell migration and during changes in the migratory and metabolic state of the cell; and (3) coding sequence coupled to arginylation constitute a novel fundamental mechanism that regulates homologous protein isoforms involved in cell migration. Longer-term, our work will extend our cell migration- related studies into a global concept for future studies that will determine the role of coding sequence and arginylation in global protein regulation.